1. Field of the Invention
This invention relates to a method and apparatus for the analysis of particles in liquid samples, and also to flow cells and other devices for use in the method and apparatus. Particularly the invention is concerned with method and apparatus in which the liquid sample is passed by liquid flow along a detection passage past a detector, for example, a detector using a laser beam. One particular application of the invention is in the analysis of blood cells by counting them to establish their concentration in a particular sample, and the discussion which follows in this specification will mainly be concerned with this use. However, the invention is not limited to this use, and other analyses may be carried out, as mentioned below.
2. Description of the Prior Art
Medical diagnosis requires analysis of blood samples to determine the blood cell concentration, for example, the white blood cell content. This is done by counting the number of blood cells in a sample. The analyzed sample may be a liquid prepared by treatment, including dilution, of the blood. One known form of blood cell analyzer uses an image processing technique, in which cells in a photograph of the sample are recognised and counted. Although this technique is precise, it is slow.
Recently, new blood cell analyzers using a flow technique have been developed. The sample is passed quickly as a thin stream through a laser detector, which counts the cells. Analysis is very quick, for example 10,000 cells per second. Two forms of detection of the cells are used, one based on the scattered light whose intensity depends on the cell size, and the other using fluorescence of a dye which is excited by the laser. The intensity depends upon the cell type.
Some devices using this flow technique are on the market, and one is disclosed in JP-A-60-97241. In these known devices, the flow in the capillary detection passage past the detector is of the type known as sheath flow, in which a carrying liquid which undergoes laminar flow and is, for example, saline solution, entrains the sample as a thin stream, which may even be discontinuous, in the carrying liquid. The sample emerges from a nozzle at a central region of a flow passage for the carrying liquid which narrows towards the detection region. Thus the speed of the carrying liquid increases, and the entrained sample stream becomes thinner. Typically, the width of the stream exiting from the nozzle is of the order of 200 microns, and at the capillary detection region the width of the stream is 10 to 20 microns. The width of the capillary itself may typically be 300 microns square. Thus in the detection region, in the case of a blood sample, the blood cells pass one by one.
The present applicants have developed new forms of sheath flow cells for use in such detection apparatus, employing microfabrication techniques disclosed in EP-A-286088, EP-A-288029 and EP-A-294701. In these flow cells the detection passage has a flat shape, rather than a square cross section, but the same principle of a thin sample stream in laminar flow applies. These new sheath flow cells have particularly low pressure drop, and are further discussed in the paper "Flat flow chambers with low pressure loss" given at the FLUCOME Conference, Sheffield, UK, 1988, by H. Ohki, R. Miyake, I. Yamazaki and T. Kaneko.
The present invention is concerned with the supply of the sample to the flow cell and certain aspects of the design of the flow cell, and is applicable to the flow cells of the prior art discussed above. The principles of blood cell analysis, and other kinds of analysis, and of sheath flow are already known and need not be described in detail here.
In the apparatus shown in JP-A-60-97241, a blood sample of predetermined amount is given a pretreatment involving dilution to make it suitable as a sample fluid for analysis. Other pretreatments may be carried out, for example, mixing with hemolysis solution and dyeing solution. After such treatment, the sample is passed through fixed tubes to the flow cell at which the analysis by detection of the cells is carried out. A blood sample may be split into two samples for analysis, one for the counting of the red blood cells and the other for the counting of the white blood cells. The requirements of this pretreatment process mean that the tubing and its associated valves and pumps is complicated, and the length of the flow path for the sample before it reaches the detector is relatively long.
Of course, the blood samples from different patients must be kept rigorously separate and the parts of the apparatus contacted by a sample must be washed between analysis of different samples. In the known apparatus, a sample is conveyed from the last pretreatment stage to the flow cell along the tubing by holding the front and rear ends of the sample with transport fluid. Inevitably, diffusion occurs at the front and rear boundaries of the sample, so that considerable time is required for a uniform concentration of the sample to reach the detector. This reduces the rate at which samples can be processed.
A second problem arises from the need to keep the samples separate in the apparatus, and to wash the parts of the apparatus contacted by the samples. In the known apparatus, a single sample is subjected to the dilution process, in a dilution tank, and therefore the processing speed may be determined by the time taken for the dilution treatment in the tank and the subsequent washing of the tank with clean fluid.